Vaccines are widely viewed as cost-effective interventions to prevent and control classical endemic and epidemic infectious diseases, as well as to limit the transmission and impact of emerging infections and certain bioterror agents. However, the development of new and improved vaccines against some of these agents is hampered by a lack of information concerning the true (i.e., operative) immunological mechanisms underlying the protection elicited by natural infection and by candidate vaccines. This is particularly true with regard to pathogens that enter the host via mucosal surfaces, including the gastrointestinal (Gl) tract. Thus, this proposal is focused on furthering our understanding of the protective immunological mechanisms that can be elicited in the Gl microenvironment of humans. Moreover, because the normal Gl flora (microbiota) is certain to influence the host immune response, we propose to conduct pioneering studies on the interactions between the local intestinal microbiota and the host immune response in humans. We will focus our efforts on in-depth studies of 3 major gram negative bacterial human pathogens including Helicobacter pylori, Salmonella enterica serovar Typhi and Shigella, each primarily affecting a distinct major segment of the Gl tract (i.e., the stomach, ileum and colon, respectively). Because virtually all of the limited information available concerning the determinants of protective mucosal immunity comes from studies in adults, we will also focus some efforts in exploring the responses to oral immunization with the licensed Ty21a typhoid in children and, for the first time, the elderly. To address the complexity of this undertaking, we have assembled a multidisciplinary team consisting of renowned investigators in the fields of innate and adaptive immunity, molecular biology, mucosal biology and physiology, biochemistry, high-throughput technology, microbiology, genomics, protein chemistry and clinical gastroenterology and vaccinology (with extensive experience in performing endoscopies and in conducting vaccine trials). In addition, we propose to develop two novel technologies to broadly advance the study of human immunology, including a human-based approach to study the entire S. Typhi ORFeome to identify CD8+ T cell responses and a peptide conformation constrainment technology and potential mucosal adjuvants to advance H. pylori vaccines. We expect this CCHI to yield much needed information in an area of great importance to human health. RELEVANCE: In spite of the great need for new and improved vaccines against major human pathogens, particularly those that enter the human host through the intestinal mucosa, this field is impeded by insufficient knowledge of the determinants of protective gut immunity. Moreover, little is known concerning the role of the gut microbiota in modulating the immunogenicity of oral vaccines and vice versa. The wealth of data generated by these studies is likely to lead to major advances in mucosal vaccine development in humans. PROJECT 1: PROTECTIVE IMMUNE MECHANISMS TO S. DYSENTERIAE 1 VACCINES IN CYNOMOLGUS MACAQUES AND HUMANS (Sztein, M) PROJECT 1 DESCRIPTION (provided by applicant): Shigella is a global infection that is notorious for disseminating rapidly in certain settings. One serotype, Shigella dysenteriae type 1 (S. dysenteriae 1), can cause devastating pandemics with high case fatality rates and thus it has been classified as a Category B priority pathogen with high potential to be used as a biological weapon. There is no available vaccine for Shigella. The development of effective Shigella vaccines has been hampered by a considerable lack of information of the specific determinants of protective immunity to Shigella infection. Because of the limitations imposed by the risks associated with performing challenge studies with wild type S. dysenteriae 1 in clinical trials to advance vaccine development, a nonhuman primate model is urgently needed. We have already established a challenge model with wild-type S. dysenteriae 1 strain 1617 which, to date, exhibited an attack rate of 100% (6 of 6 cynomolgus macaques challenged with 10e11 cfu intragastrically). Furthermore, we have advanced our understanding of the immune responses elicited following challenge. In this application we propose to continue these studies by addressing the following Specific Aims: (1) evaluate the hypothesis that intragastric immunization with novel attenuated S. dysenteriae 1 mutant strains elicits protection from intragastric challenge with wild type S. dysenteriae 1; (2) evaluate the hypotheses that a defined set of immune responses observed in circulation in cynomolgus immunized with attenuated strains of S. dysenteriae 1 and/or challenged with wild type S. dysenteriae 1 correlate with protection and are representative of those present at effector sites (i.e., mucosal tissues) and secondary lymphoid organs. These translational studies are central to further our understanding of the immunological mechanisms that mediate protection to S. dysenteriae 1 and longevity of the responses to vaccination in humans, (3) To evaluate the effects of immunization of monkeys with attenuated S. dysenteriae 1 strains on the colonic microbiota in stools of monkeys and the impact of the existing microbiota on the observed immune responses and protection from challenge. Finally, we will take advantage of an upcoming trial with the attenuated S. dysenteriae 1 strain CVD 1256 to evaluate the hypothesis that the immune responses observed systemically and locally in humans are similar to those that correlate with protection in cynomolgus macaques (Aims 1 and 2). These studies will provide valuable insights that might accelerate the development of attenuated vaccines for S. dysenteriae 1. RELEVANCE: The overall objective of this project is to develop a safe and effective vaccine for S. dysenteriae 1, a Category B priority pathogen with potential to be used as a biological weapon. Currently, there is no available vaccine for Shigella and limited treatment options for infections with multiple antibiotic resistant strains. Given the shortcomings of available measures to successfully control this infection, and its bioterrorism potential, to develop a S. dysenteriae type 1 vaccine is of great importance.